Protein Primary Structure of the Vaccinia Virion at Increased Resolution

Abstract

Here we examine the protein covalent structure of the vaccinia virus virion. Within two virion preparations, >88% of the theoretical vaccinia virus-encoded proteome was detected with high confidence, including the first detection of products from 27 open reading frames (ORFs) previously designated "predicted," "uncharacterized," "inferred," or "hypothetical" polypeptides containing as few as 39 amino acids (aa) and six proteins whose detection required nontryptic proteolysis. We also detected the expression of four short ORFs, each of which was located within an ORF ("ORF-within-ORF"), including one not previously recognized or known to be expressed. Using quantitative mass spectrometry (MS), between 58 and 74 proteins were determined to be packaged. A total of 63 host proteins were also identified as candidates for packaging. Evidence is provided that some portion of virion proteins are "nicked" via a combination of endoproteolysis and concerted exoproteolysis in a manner, and at sites, IMPORTANCEPoxviruses are among the most complex and irregular virions, about whose internal structure little is known. To better understand poxvirus virion structure, imaging should be supplemented with other tools. Here, we provide a deep study of the covalent structure of the vaccinia virus virion using the various tools of contemporary mass spectrometry. C ontemporary mass spectrometry (MS) instrumentation provides an avenue for proteome coverage with enormous breadth or depth, depending upon the complexity of the proteome. Typically, the power of the approach is exploited for breadth in the analysis of increasingly complex proteomes. Here, we have taken a relatively narrow proteome, that of a virus particle, and explored proteome depth, developing analytical approaches and tools as required.Sporadic information is available on the covalent (primary) molecular structure of the vaccinia virus virion. Around 75 proteins can be detected in two-dimensional (2D) gels of purified vaccinia virus virions (1). Three published MS studies provided early compendia of viral and host gene products present within vaccinia virus and human monkeypox virion preparations via MS (2-5). These studies built upon earlier, less comprehensive studies that employed a combination of MS and N-terminal sequencing (1, 6). Overlapping approaches in terms of instrumentation, preparation, and data analysis led to an overlapping set of detected gene products.In terms of covalent protein modifications, six vaccinia virus proteins can be labeled in vivo with myristate, five of which have been identified as L1R (7,8), G9R, A16L, and E7R (9), and A14L (10). All except A14L possess a glycine at position 2 in the protein.At least eight vaccinia virus-encoded proteins may be palmitoylated, as evidenced by the incorporation of 3 H-labeled palmitate in culture (11). These proteins include wrapped virus (WV)-specific protein p37 (F13L) (7,(12)(13)(14) and products of open reading frames (ORFs) A33R (15), B5R (16), F13L (17), the A22R Holliday resolvase (11), A...

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